Bacterial Expression Vectors

Inducible protein expression systems for Gene Synthesis orders or from the ATUM Catalog.

T5

T7

Rhamnose

Arabinose

PhoA

Expression

Inducible

Inducible

Inducible and Titratable

Inducible and Titratable

Inducible

Expression Tuning

RBS choice

RBS choice

[Rha] and RBS choice

[Ara] and [IPTG]

RBS choice

Localization

Intracellular or Secreted

Intracellular

Intracellular or Secreted

Intracellular

Intracellular

IP

IP-Free

IP-Free

IP-Free

IP-Free

IP-Free

Recommended Vector Selection Process

1. Single Expression VectorAlready know exactly which combination of elements you want? Use the Vector Selector to choose one of our ready-to-use vectors.

2. Expression PanelNot sure how your protein will behave? Test your gene in several vectors with a variety of properties most likely to increase expression. ATUM offers pre-selected Expression or Secretion panels, or you can create your own to explore the properties you think are most important.

3. VectorGPS®Want the very best vector for your system? ATUM will create unique combinations and configurations of vector components using our machine learning technology, to create a custom vector exactly suited to your needs.

Protein Expression from Different Promoters

Different promoters work better for different proteins. Expression of three proteins, phi29 (~61 kD), cutinase (~22 kD) and DasherGFP (~26 kD), under control of different promoters, T5 and T7 (IPTG-inducible), rham (rhamnose-inducible), phoA (inducible by phosphate starvation), and ara (arabinose-inducible, only DasherGFP expression tested) is shown. Expression values shown on the y-axis are measurements of expressed protein band densities from a SDS-PAGE gel using BSA as standard.*DasherGFP measured fluorescence is lower with T7 and rham expressed GFP, indicating that a fraction of total protein is incompletely folded protein.

Use the ATUM Expression Test to determine the best vector for your construct. ATUM will clone and test your construct in a full bacterial vector panel to quickly determine expression levels in E. coli.

Independent Expression Using Two Orthogonal Promoters

Choice of promoters and inducible systems allow orthogonal expression of two proteins in a single strain by using a two vector system for induction control of each individual protein. Choose from vectors with promoters that are IPTG-inducible (T5 or T7), rhamnose-inducible, arabinose-inducible, or inducible phoA.

Orthogonal Expression of YFP and CFP in a Two Vector System:

Cloning of YFP and CFP into two pDAUGHTER vectors – rham_YFP and T5_CFP

No interference observed – expression is exclusive

Comparable expression with Rham and T5 promoters

Orthogonal Expression in a Single Strain

Fluorescence of KringleYFP in pD8xx (pD884-12C, low copy) was measured at excitation/emission of 520/540 nm to exclude any background signal from CFP. This gives a better signal-to-noise ratio.

Fluorescence of CindyLouCFP in pD444 was measured at excitation/emission of 395/500 nm.

Vector Cost

Vector Cost for Gene Synthesis Projects: Cloning of synthetic genes into the first vector is available free of charge. If the same gene is cloned into multiple vectors, additional fees will apply. Regular gene synthesis fees apply.

ATUM can even test your gene for high expression.Expression Test is now available for bacterial vector panels with a volume discount. Cost for expression testing is $395.00 USD per gene per vector with an additional 10% off the total.
Prices are in addition to regular gene synthesis fees.

T7 Promoter: The T7 promoter results in high protein expression levels and is repressible. The T7 promoter only works in T7 pol expressing E. coli strains (eg. BL21(DE3) or T7 Express). Note that strains in which basal expression is reduced, such as those carrying lysS or lysY, frequently express at lower levels after induction when compared with strains that carry only the T7 polymerase gene.

PhoA Promoter: The bacterial alkaline phosphatase (phoA) promoter is a strong promoter that is inexpensively induced when the culture is starved for inorganic phosphate. In the presence of phosphate, expression is tightly regulated making this system useful for expression of toxic proteins. Very high product yields have been obtained by use of the phoA system, for example gram per liter yields of an active Fab have been achieved using this system. Other examples of proteins expressed using this system are hEGF and HPRT where high yields of soluble protein were obtained.

Arabinose Promoter: The arabinose-inducible promoter ara is capable of high level recombinant protein expression in the presence of arabinose and is tightly regulated by glucose in the absence of arabinose. The ara promoter controls the genes ara organized in one operon. The promoter is flanked by a pair of lac operators that are recognized by the lac repressor also carried on the plasmid. IPTG binds to the repressor, inducing expression. The ara promoter is compatible with E. coli strains BL21 or DH5α.

Rhamnose Promoter: Rhamnose-inducible bacterial expression vectors with different strength ribosome binding sites and origins of replication provide an excellent range of induced and uninduced expression levels. Rhamnose Expression Vectors are tightly controlled by rhamnose, enabling high expression yields, even for toxic or challenging proteins and can be used in any E. coli strain or other Gram-negative bacteria.Rhamnose Induction protocol for pD8xx vectors.

Rhamnose FAQ:

Is the Rhamnose promoter stronger than the T5 promoter?
At high rhamnose concentrations, we are seeing similar amounts of protein to T5. However, T5 might be somewhat stronger.

At what phase do you induce with Rhamnose?
For standard applications we induce by adding rhamnose at mid-log just as we do with IPTG.

Is induction by Rhamnose completely repressed by glucose?
In E. coli, glucose shuts down expression very well, even in the presence of rhamnose. This is why it works for autoinduction. You add both rhamnose and glucose, once glucose is consumed rhamnose induces.

Do ATUM rhamnose vectors carry the rhaRS gene; and if not, does that affect expression in rhaRS+ strains (e.g. wild type E. coli)?
No, our rhamnose vectors do not carry the rhaRS gene and it does not affect expression in wild type E. coli.

Vector Selector

Overview

Solubility-enhancing tags are generally large peptides or proteins that increase the expression and solubility of fusion proteins. No single fusion tag can increase the expression and solubility of all target proteins. Therefore, ATUM offers vectors with a variety of solubility tags. A comparison of fusion tags that help improve solubility of some difficult to express soluble proteins is shown below. Select a tag from the interactive graph or get the entire set to determine the best tag for your protein.

Effectiveness of Solubility Tags is Protein Dependent

Expression of total and soluble protein obtained using different tags. Three proteins encoded by genes A (~48 kD), B (~49 kD) and C (~59 kD), having shown poor solubility, were expressed in IPTG-inducible T5 promoter E. coli expression vectors (pD441-XXX) with various N-terminal solubility tags: MBP, GST, PpiB, and Fh8; 6XHis was included as a negative control. Cultures were grown at room temperature (RT) and 37°C. Overall, higher soluble protein expression was observed at RT (data shown). Percent solubility is shown along the Y-axis and the size of the circles corresponds to protein yield shown in nmole/µl. Legend shows approximate yield of protein based on circle size.

An integral part of tag choice is the method for removing the tag after purification. This step almost always involves using a protease to cleave a specific peptide bond between the tag and recombinant protein. ATUM offers the tobacco etch virus (TEV) protease with a 6XHis tag and the HRV3C protease with a GST tag to facilitate removal of the enzyme post-cleavage.

Use the ATUM Expression Test to determine the best vector for your construct. ATUM will clone and test your construct in a full bacterial vector panel to quickly determine expression levels in E. coli.

Notes

FH8 solubility tag is licensed from Hitag and is available for research use only. A separate license is required for any commercial use, including the development of commercial products or services. Information about commercial licenses may be obtained from Hitag Biotechnology. Lda, Biocant Park L4 N4, 3060-197, Cantanhede, Portugal.

Vector Selector

Overview

Affinity tags are known epitopes that when fused to either the C- or N-terminus of a recombinant protein are highly efficient tools for purifying proteins from crude extracts. This enables highly selective capture and circumvents multistep purification processes. In addition, the tags can be used for detection and may also help enhance protein expression.

N-terminal fusions can also help with expression as they can iron out any secondary structure that interferes with expression.

An integral part of fusion tag choice is the method for removing the tag after purification. This step almost always involves using a protease to cleave a specific peptide bond between the tag and recombinant protein. ATUM offers the tobacco etch virus (TEV) protease with a 6XHis tag and the HRV3C protease with a GST tag to facilitate removal of the enzyme post-cleavage.

Vector Selector

Overview

ATUM vectors with different secretion signals allow selection of the secretion signal best suited to each protein. Parallel processing of multiple pDAUGHTER secretion vectors allows a quick and convenient method for selecting the signal(s) which provides the highest levels of secreted expression. While different secretion signals have been used, individual levels of secretion may vary and depend upon the recombinant protein being expressed. There is no general rule to selecting a secretion signal to guarantee successful secretion for a given recombinant protein, as efficiency of secretion depends on host strain, signal sequence and type of protein.

Different proteins prefer different secretion signals. Periplasmic expression of MBP, Cutinase, and Alkaline phosphatase from rhamnose vectors with different secretion signals. Total cellular protein levels are represented as circle diameter. Soluble protein in the periplasm is shown on the y-axis. Protein was quantified by densitometry of stained acrylamide gels.

Use the ATUM Expression Test to determine the best vector for your construct. ATUM will clone and test your construct in a full bacterial vector panel to quickly determine expression levels in E. coli.

Why secreted expression?

Production of secreted recombinant proteins using E. coli rhaBAD promoters offers several advantages compared to cytosolic production, such as simplicity of purification, avoidance of protease attack and N-terminal Met extension, and a better chance of correct protein folding. Since the use of a post-translational or co-translational export mechanism is protein specific and cannot be known a priori, vectors with different secretion signals allows for the selection of the translocation pathway best suited for a given recombinant protein.

Note: ATUM recommends the use of vectors with rhamnose inducible promoters for secreted protein expression. There are many distinct advantages to the rhamnose secretion vectors:

Expression of protein is tightly tunable with rhamnose concentration thus allowing you to modulate protein expression, so as to avoid overwhelming the secretion machinery.

Rhamnose vectors are available with a wider selection of 18 secretion signals spanning the Sec, TAT, and SRP translocation pathways.